At 05:09 PM 7/10/97 +0100, Kris W. wrote:
>The other day I was digesting a PCR-Script vector with EcoRI and I found
>that it cut at two sites when I was only expecting it to cut at one. I
>normally leave the digestion for an hour or so but this time I left it
>for 3 hours. I re-did the digestion for a half hour and found that
>there was only a single cut. After looking at the sequence and the
>sizes of the fractions caused by the digestion I concluded that EcoRI
>(gaattc) cuts at aaattc when left for too long. Similar sites such as
>gaattg were not cut.
>>Has anyone else noticed this or similar incidences with other
>restriction enzymes.
>>
What you describe is nothing unusual for some enzymes (eg. EcoRI, SalI etc.). It has been known as "Star Activity" for a couple of decades and every student of molecular biology has had an encounter with this (or, will have). In such cases the recognition sequence for the RE in question is reduced by one/two base(s) when the digestion is carried out (i) for more than an hour, (ii) with too much glycerol (>5%)or (iii) in very low-salt buffers etc. or under a combination of these conditions. The rule of thumb in such cases is to increase the salt concentration to at least 150mM in the mix if the digestion is to be done longer than an hour and to make sure that the enzyme volume does not exceed 10% of the reaction volume (enzymes come in 50% glycerol). Keep in mind that the sp. activities of these enzymes these days are at least 10u/uL. And I hope you know what that means.
The "Star Activity" has been explained also in a number of molecular biology product handbooks and the ones that come to mind are the New England Biolab and the Pharmacia catalogs.
Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu